Hematology Analyzer and Quality Control Khaimuk Changsri
Automated CBC red blood cell analysis - Count - RBC indices - Hematocrit / Hemoglobin conc. - Size and shape - RBC clumping Platelet cell analysis - Count - PLT indices - PLT-crit - PLT clumping Leukocyte analysis - Count - Differntial (Neutrophil, Eosinophil,Lymphocyte, Monocyte) Extra Diff : nucleated RBC, Blast Option: abnormal WBC (atypical lymphocyte)
TYPE of AUTOMATE analyzer • a three-parts diff(granulocytes, lymphocytes, and Monocyte/mix cell differentiate) • a five-parts diff (neutrophils, lymphocytes, monocytes, eosinophil's, and basophils. • a seven parts diff ( 5-parts diff + 1. nucleated RBC (NRCs) 2. Abnormal and atypical Lymphocyte, Immature cells )
ADVANTAGES • SPEED WITH • ACCURACY • ABILITY TO • SIGNIFICANT • INVALUABLE EFFICIENT AND PRECISION PERFORM FOR ACCURATE MULTIPLE REDUCTION OF DETERMINATIO HANDLING OF IN TESTS ON A N OF RED CELL LARGE NUMBER QUANTITATIVE SINGLE LABOR BLOOD TESTS. PLATFORM. INDICES. OF SAMPLES. REQUIREMENTS.
General Principles • Introduction to Instrumentation principles used : • Electronic Impedance • conductivity (radio frequency) • Optical Scatter a. Laser light scatter b. Flow cytometry c. Chemical dye method
Electrical Impedance Cell count Cell volume 1st The Coulter Principle
OPTICAL SCATTER
Low angle scatter 2o - 3o High angle scatter 5o - 15o Forward-angle light scatter (FALS) - size , volume of Cell Side scatter (SSC) • 90o -a granularity signal or complexity of Cell
Conductivity
Peroxide based: Myeloperoxidase enzyme (MPO) is used to count neutrophils. Lymphocytes not stained Fluorescence based: Retic and platelet count. Immunological based: Immature platelets -Accurate platelet count using CD41/CD61 antibodies
Hemoglobin Estimation Lyse treated blood • Cyanide Solution added Cyanmeth hemoglobin method • Sodium Lauryl Sulphate added Sulphmeth homoglobin method
Lyse reagent lysed dilution Hgb Flow Cell impedance method Lyse reagent : destroy RBC Hemoglobin PLT - Particles between WBC's membrane collapse concentration around the nucleus 2 and 20 fL Calculated Hct, MCH, MCHC • HCT(%) = MCV x RBC RBC - > 36 fL • MCH(pg) = Hb / RBC • MCHC(g/dl) = Hb / HCT RBC count WBC count > 30 fL 3 parts diffs MCV Lymphocyte – 35-90 fL RDW Granulocyte – 150-450 fL PLT count Mono nuclear cells (mono, 2-30 fL eo,baso) 90-160 fL
The Coulter Principle VCS Technology (Volume, Conductivity, and Scatter)
VCS technology (DC) Impedance RFc Laser VOLUME - (DC) Impedance in isotonic diluent. CONDUCTIVITY the radio frequency (RF) penetrate the cell. - cell size and internal structure, including chemical composition and nuclear volume. LIGHT SCATTER ,LASER beam, the scattered light spreads out in all directions. -median angle light scatter (MALS) signals, -cellular granularity, nuclear lobularity and cell surface structure
[Y] 3D- cellular Analysis Volume DC [Z] Conductivity RF [X] Light Scatter
Normal data plot
DOT plot or Scatter gram Differential Chanel WBC /Basophil Chanel
Optical Differential Sysmex XE5000 DCxRF
Advia 2120i White Blood Cell Technology Dual WBC counts and Differentials. Optical Light Scatter. Peroxidase Staining. CellSize VS Peroxidase Activity Nuclear complexity VS CellSize
Advia 2120i Red Blood Cell Cytogram Visual analysis Red Blood Cell Technology Cellular Hemoglobin VS Volume of rbc’s.
Advia 2120i Platelet Analysis
Hematological analyzer analysis example.
Normal
Neutrophilic granulocytosis
Lymphocytosis
Basocytosis with left shift
Analysis Leukopenia Thombocytopenia microcytosis
NEW PARAMETERS • Nucleated RBCs • Immature granulocyte • Hematopoitic progenitor cells • Immature reticulocyte fraction • RBC fragments (schistoytes) • Reticulated platelets • Reticulocyte indics • Malarial parasites
NEW PARAMETERS Cellular Hb Concentration Mean(CHCM): Uses Light scatter technology. True estimate of hypochromia in IDA. • Hb Distribution Width: Degree of variation in red cell hemoglobinization. Range-1.82 to 2.64. • Nucleated Red Cells: fluorescence & forward scatter.
Newer Parameters(contd..) Reticuloctes: Various dyes & flurochromes bind with RNA RNA content- 3 Maturation stages; LFR,MFR & HFR Immature reticulocyte Fraction(IRF): Sum of MFR & HFR. Early and sensitive index for erythropoisis. Reticulocyte Hb Equivalent(RET-He): Hb content of freshly prepared RBCs. Real time information on Fe supply to erythropoiesis. Early detection of Fe deficiency. Differentiate IDA & ACD. Monitoring of erythropoietin & Fe therapy.
malaria ✓ Malaria found ✓ Variable and large lymphocyte and monocyte
Reticulated Platelets /Immature Platelet Fraction(IPF): Newly produced platelets that have remains of RNA in their cytoplasm. Reflects rate of thrombopoiesis
Instrument and generation Technology manufacturer Coulter, STKS, GEN-S, Bekman-Coulter LH 700 series VCS technology (volume, conductivity, and Scatter Sysmex SE series, XE 2100 1. Impedance with low-frequency Instrumentation XS, XT electromagnetic current XN SERIES 2. Impedance with high-frequency electromagnetic current 3. Laser light scattering 1. Impedance with low-frequency direct current 2. Impedance with radiofrequency current 3. Hydrodynamic focusing Fluorescent light scattering , 3
Instrument and generation Technology manufacturer Cell-Dyn 1800 VCS technology (volume, conductivity, Cell-Dyn and Scatter Technology Cell-Dyn Cell-Dyn 3500, 3700 Multiple-Angle Polarized Scatter Technology Separation (MAPSS) 1. Four light-scattering parameters: forward light scatter, orthogonal light scatter, narrow-angle light scatter, and depolarized orthogonal light scatter 2. Hydrodynamic focusing 3. Use of flow cells Ruby, Rapphire Above + fluorescence
3 parts differential
Lyse reagent lysed dilution Hgb Flow Cell impedance method Lyse reagent : destroy RBC Hemoglobin PLT - Particles between WBC's membrane collapse concentration around the nucleus 2 and 20 fL Calculated Hct, MCH, MCHC • HCT(%) = MCV x RBC RBC - > 36 fL • MCH(pg) = Hb / RBC • MCHC(g/dl) = Hb / HCT RBC count WBC count > 30 fL 3 parts diffs MCV Lymphocyte – 35-90 fL RDW Granulocyte – 150-450 fL PLT count Mono nuclear cells (mono, 2-30 fL eo,baso) 90-160 fL
PLT-RBC Size distribution curve DW = abnormal distribution width Same RL or RU RDW-SD,RDW- CV flaged 20% of Hight RL = abnormal height at RBC LD RU = abnormal height at RBC LD MP = multiple peak of RBC histogram Giant Platelet Cold agglutination Extreme anisocytosis is found Micro-Erythrocyte RBC agglutination Dimorphic picture Platelet clumping Rouleaux formation Note: Parameter: RBC, MCV, RDW-SD Dysplastic RBC (small/fragmented) & RDW-CV are flagged.
RDW-CV and RDW-SD = marker for anisocytosis L1 L2
Mean corpuscular volumn Right shift Macrocytic anemia Left shift Microcytic anemia
PLT histogram / Thrombocytic histogram PLT counted 2 fl -30 fl. flexible Discriminator PL 2 to 6 fl. flexible Discriminator PU 12-30 fl. Parameter of the thrombocyte P-LCR MPV 8-12 fL = ratio of large platelets 15 - 35 % PLT clumping Giant PLT Microcyte Fragmented RBC PDW PDW = platelet distribution width calculated at 20 % of peak height Reference
Thrombocyte histogram Flagging “ MP “, multiple peaks “ PH “, ab height at HD LD HD Cause: Platelet anisocytosis , Recovery after chemotherapy, PLT Platelet aggregation 2 10 20 30 size (fL) “ PL “, ab height at LD PLT_C: incorrect high cause: High blank value /Cell Case :platelet aggregation fragments / High numbers of bacteria RBC fragmented , microcyte , giant plt background check
“ PH “, ab height at HD Plt aggregation False low Plt count Check tube?clot? / correct ratio of EDTA giant platelets False low Plt count or recollect blood with Microerythrocytes False High Sodium citrate Fragmentocytes or False High dysplastic RBC Manually count with counting chamber / smear Observed from MCV / blood smear : resolved by PLT estimation Observed from RDW / blood smear : resolved by PLT estimation
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